Compositions containing legume products

ABSTRACT

The present invention features legume products having trypsin inhibitory activity and reduced microbial content, methods of decontaminating such legume products, compositions containing such legume products, and the topical application of such legume products or compositions to skin, nails, and hair.

This patent application is a divisional of prior application Ser. No.09/796,054, filed Feb. 28, 2001 now U.S. Pat. No. 7,192,615 and herebyincorporates said patent application by reference herein.

FIELD OF THE INVENTION

The present invention relates to legume products, topical compositionscontaining such legume products, and the manufacture and use thereof.

BACKGROUND OF THE INVENTION

Legume fruits contain high levels of proteins, lipids and carbohydrates.Consequently, legume fruits, such as soybeans, and compositionscontaining legume fruits are considered a great nutrient for human use.Legume fruits also contain compounds that inhibit protease activity. Forexample, two protein protease inhibitors were isolated from soybeans inthe early 1940's, the Kunitz-type trypsin inhibitor (soybean trypsininhibitor, STI) and the Bowman-Birk protease inhibitor (BBI). See, e.g.,Birk, Int. J. Pept. Protein Res. 25:113-131 (1985) and Kennedy, Am. J.Clin. Neutr. 68:1406S-1412S (1998).

STI inhibits the proteolytic activity of trypsin by the formation of astable stoichiometric complex. See, e.g., Liu, K., Chemistry andNutritional value of soybean components. In: Soybeans, chemistry,technology and utilization. pp. 32-35 (Aspen publishers, Inc.,Gaithersburg, Md., 1999). STI consists of 181 amino acid residues withtwo disulfide bridges and is roughly spherically shaped. See, e.g., Songet al., J. Mol. Biol. 275:347-63 (1998).

BBI is an 8 k-Da protein that inhibits the proteases trypsin andchymotrypsin at separate reactive sites. See, e.g., Billings et al.,Pro. Natl. Acad. Sci. 89:3120-3124 (1992). STI and BBI are found only inthe soybean seed, and not in any other part of the plant. See, e.g.,Birk, Int. J. Pept. Protein Res. 25:113-131 (1985).

However, due to its natural origin, high levels of microorganisms arecarried on the outside of legume IC fruits, such as soybeans.Consequently, decontamination processes such as heat, organic/aqueoussolvent extraction, and high shear purification may be used to reducesuch microorganism concentrations to allow it to be safe for human use,e.g., skin care applications. Applicants, however, have found that theseprocesses, which frequently denature the active compounds in the soy,result in a compromised biological efficacy (e.g., a reduction inprotease inhibitory activity) which is important for cosmetic andtherapeutic uses to the skin, hair, and nails. Furthermore, suchprocesses also can lead to instability of the soy product as well as toan undesirable odor and color generation. Therefore, there is acommercial need to develop a means to reduce the levels of microbials insoy products without compromising the biological benefits of suchproducts.

The object of the present invention is to provide for a soy product(e.g., that can be used as an ingredient in a skin, hair, or nail carecomposition) that has reduced microbial content but maintains itsprotease inhibitory activity. Another object of the invention is toprovided for a skin, hair, or nail care composition containing such soyproduct optionally with other active agents.

The present invention relates to legume products containing reducedmicrobial content that retains legume's beneficial biologicalactivities, processes for obtaining such legume products, and usesthereof in cosmetic compositions.

SUMMARY OF THE INVENTION

The present invention features legume products having trypsin inhibitoryactivity and reduced microbial content, methods of decontaminating suchsoy products, and compositions containing such soy products. In onepreferred embodiment, the legume product is a soy product.

The present invention also relates to the topical application of legumeproducts or compositions containing such legume products for use in themaintenance of healthy skin, nails, and hair as well as the preventionor the treatment of skin, nails, and hair disorders, including, but notlimited to: regulating firmness of the skin, hair, or nails; cleansingthe skin, hair or nails; reducing and/or delaying hair or nail growth;straightening and/or lightening of hair; treatment and/or prevention ofacne; regulating the tone of skin, hair, or nails; regulating thetexture of skin, hair, or nails; regulating wrinkles in skin; treatmentof external aggressions in skin, hair, or nails; and beautifying theskin, hair, or nails.

Other features and advantages of the present invention will be apparentfrom the detailed description of the invention and from the claims

DETAILED DESCRIPTION OF THE INVENTION

It is believed that one skilled in the art can, based upon thedescription herein, utilize the present invention to its fullest extent.The following specific embodiments are to be construed as merelyillustrative, and not limitative of the remainder of the disclosure inany way whatsoever.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. Also, all publications, patentapplications, patents, and other references mentioned herein areincorporated by reference. As used herein, all percentages are by weightunless otherwise specified.

As used herein, “trypsin inhibitory activity” means the ability of thelegume product at a concentration of 0.1% (w/w) to inhibit the activityof the protease trypsin, as measured by the assay set forth below inExample 2. In one embodiment, the legume products of the presentinvention have a trypsin inhibitory activity of at least about 15%. In afurther embodiment, the legume products of the present invention have atrypsin inhibitory activity of at least about 25%, such as at leastabout 50%.

As used herein, “thiol retention activity” means the ability of thelegume product at a concentration of 1% (w/v) to inhibit smoke-inducedloss of thiols, as measured by the assay set forth below in Example 3.In one embodiment, the legume products of the present invention have athiol retention activity of at least about 75%. In a further embodiment,the legume products of the present invention have an thiol retentionactivity of at least about 90%, such as at least about 95%.

As used herein, “microbial content” means the amount of bacteria, fungi,and yeast present in the legume product Examples of means to measuremicrobial content include, but are not limited to, the AOAC 986.23Method as set forth in “Official Methods of Analysis of AOACInternational,” edited by Patricia Cunniff, Sixteenth Edition, 5^(th)Pevision, 1999 (AOAC International) or the USP Method as set forth in ha“Official Compendia of Standards, USP 24 USP/NF 19”, United StatesPharmacopeial Convention, Inc., 2000 (Board of Trustees, United StatesPharmacopeial Convention, Inc.).

“Objectionable microbial content” means the amount of bacteria, fungi,and yeast present in the legume product that are harmful to humans,including but not limited to coliform, E. Coli, Salmonella, thermophilicspores, Bacillus, Enterococcus, Staphylococcus, fecal streptococcus, andthose listed in “Disinfection, sterilization, and preservation” 4thedition, Seymour S. Block, pp. 887-888 (1991, Lea & Febiger, Malvern,Pa.)

As used herein, “topical application” means directly laying on orspreading on outer skin using, e.g., by use of the hands or anapplicator such as a wipe, puff, roller, or spray.

As used herein, “cosmetically-acceptable” means that the product(s) orcompounds) which the term describes are suitable for use in contact withtissues (e.g., the skin) without undue toxicity, incompatibility,instability, irritation, allergic response, and the like.

As used herein, “topical carrier” means one or more compatible solid orliquid filler diluents that are suitable for topical administration to amammal. Examples of topical carriers include, but are not limited to,water, waxes, oils, emollients, emulsifiers, thickening agents, gellingagents, and mixtures thereof.

As used herein, “regulating the firmness” means the enhancing of thefirmness or elasticity, preventing the loss of firmness or elasticity,or preventing or treating sagging, lax and loose skin, hair, or nails.The firmness or elasticity of the skin can be measured by use of acutometer. See Handbook of Non-invasive Methods and the Skin, eds. J.Serup & G. Jemec, Chapter 14.3 (1995). The loss of skin elasticity orfirmness may be a result of a number of factors, including but notlimited to aging, external aggressions, or the result of an applicationof a cosmetic to the skin, hair, or nails.

As used herein, “regulating the tone” means the lightening and/ordarkening of the appearance of the skin, hair, or nails (e.g.,lightening pigmented lesions, darkening skin sallowness, and/or eveningthe color of the skin).

As used herein, “delaying or reducing nail growth” means the delaying orreducing the growth rate of the nail.

As used herein, “delaying or reducing hair growth” means the delaying orreducing the growth rate of the hair and/or width of hair shaft,including, but not limited to, the reducing the visibility or appearanceof hair (e.g., hair on the arms, legs, and face).

As used herein, “cleansing” means the removal of dirt and/or oil fromthe skin, hair, or nail surface.

As used herein, “regulating the texture” means the smoothing of thesurface of the skin, hair, or nail to remove either bumps or crevasseson the surface, including, but not limited to, smoothing or evening theappearance of the skin.

As used herein, “regulating wrinkles in skin” means preventing,retarding, arresting, or reversing the process of wrinkle or fine lineformation in skin, including, but not limited to, reducing thevisibility or appearance of wrinkles.

As used herein, “treatment of external aggressions” means the reductionor prevention of the damage from external aggressions in skin, hair, ornails. Examples of external aggressions include, but are not limited to,damage to the skin, hair, and nails from the use or cleansers (e.g.,skin and hair cleansers containing surfactants), make-up, and shavingand cutting, as well as environmental damage such as from the UV light(e.g., sun damage from the sunlight or non-natural sources such as UVlamps and solar simulators), ozone, exhaust, pollution, chlorine andcompounds containing chlorine, and cigarette smoke. Effects of externalaggressions on the skin, nails, and skin include, but are not limitedto, oxidative and/or nitrosative damage to and modifications on lipids,carbohydrates, peptides, proteins, nucleic acids, and vitamins. Effectsof external aggressions also include, but are not limited to, loss ofcell viability, loss or alteration of cell functions, and changes ingene and/or protein expression.

As used herein, “safe and effective amount” means an amount of compoundor composition (e.g., the legume product) sufficient to induce apositive modification in the condition to be regulated or treated, butlow enough to avoid serious side effects. The safe and effective amountof the compound or composition will vary with the particular conditionbeing treated, the age and physical condition of the end user, theseverity of the condition being treated/prevented, the duration of thetreatment, the nature of other treatments, the specific compound orproduct/composition employed, the particular cosmetically-acceptablecarrier utilized, and like factors.

Legume Product

What is meant by a “legume product” is a substance derived from a legumefruit. A legume is a plant from the family Leguminosae, which has adescent fruit such as a bean, pea, or lentil. Examples of legumes,include but are not limited to, beans such as soybeans, lentil beans,peas, and peanuts.

The legume product may contain the entire legume fruit (e.g., the legumefruit ground into a powder) or only a portion of the legume (e.g., anextract of the legume). The legume product may be in the form of a fluid(e.g., a mixture of the legume fruit and water) or a solid (e.g., legumefruits powders). When in the form of a fluid, the term “legume product”refers to the solid constituents of the fluid derived from the legume.

The compositions of the present invention comprise a safe and effectiveamount of the legume product (e.g., soy product). In one embodiment, thecomposition contains from about 0.001% to about 50%, from about 1% toabout 30%, of the legume product (e.g., soy product).

Soy Product

What is meant by a “Soy Product” is a substance derived from thesoybean. The soy product may contain only a portion of the soybean(e.g., an extract of the soybean such as a lipid reduced soybean powderor filtered soymilk) or may contain the entire soybean (e.g., a groundpowder of the legume). The soy product may be in the form of a fluid(e.g., soymilk) or a solid (e.g., a soybean powder or soymilk powder).When in the form of a fluid, the term “soy product” refers to the solidconstituents of the fluid that are derived from the soybean.

In one embodiment, the soy product is soybean powder. Soybean powder maybe made by grinding dry soybeans. In one embodiment, the soybean powderhas a average particle size of less than about 10 micrometers such asless than about 1 micrometer in one embodiment, the soybean powder has amoisture content of less than about 10% such as less than about 5%. Inone embodiment, the soybean powder is lyophilized.

In one embodiment, the soy product is soymilk or soymilk powder. Soymilkis a combination of solids derived from soybeans and water, the mixtureof which has some or all of the insoluble constituents filtered off.Soymilk powder is evaporated soymilk, which in one embodiment, is in alyophilized or spray-dried form. Procedures for manufacturing soymilkinclude, but are not limited to, the following three procedures. First,soymilk may be made by placing soybeans into water to allow them toabsorb the water. The swelled beans are then ground and additional wateris then added. The mixture may then filtered to remove any insolubleresidue. Second, soymilk may also be prepared from soybean powder.Soybean powder is thoroughly mixed with water (e.g., for at least onehour), which may then be followed by a filtration process to removeinsoluble residues. Third, soymilk can also be reconstituted fromsoymilk powder by adding water. In one embodiment, soymilk comprisesfrom between about 1% to about 50%, by weight (e.g., from about 5% toabout 20%, by weight) of solids from the soybean.

Anti-Microbial Treatment of Legume Product

As discussed above, the surface of legume fruits often contain highlevels of microorganisms. Thus, prior to use by humans, the legumeproduct needs to be treated to reduce or eliminate such microorganisms.

In one embodiment, the legume products of the present invention have atotal microbial content of less than about 10,000 colony-forming units(“cfu”) per gram. In a further embodiment, the soy products of thepresent invention have a microbial content of less than about 1,000 cfuper gram (such as less than about 100 cfu per gram) of the legumeproduct.

In one embodiment, the legume products of the present invention have atotal objectionable microbial content of less than 300 cfu per gram suchas less than 150 cfu per gram. In a further embodiment, the legumeproducts of the present invention have an undetectable amount of anyobjectionable microbials for at least one gram (e.g., at least tengrams) of legume product.

In one embodiment, the legume product is exposed to gamma irradiation.In a further embodiment, the legume product is exposed to between about2 to about 30 kGy of gamma irradiation, such as between about 5 andabout 10 kGy of gamma irradiation. Applicants have unexpectedly foundthat such treatment reduces the microbial content of the legume product,while maintaining its biological activity (e.g., serine proteaseinhibitory activity). Applicants have also found that treatment oflegume products with gamma irradiation maintains the cosmetic eleganceof the legume product, such as maintained its natural colors and did notinduce significant malodors.

Other anti-microbial processes that also maintain the proteaseinhibitory activity of the legume product that can be practiced alone orin combination with gamma irradiation, include, but are not limited to,exposure to x-rays, high energy electron or proton beams, ultravioletradiation, hydrostatic pressure, and addition of chemical agentspossessing antimicrobial activity, and combinations thereof. A completelist of methods for microbial content reduction is set forth in“Disinfection, sterilization, and preservation” 4th edition, Seymour S.Block, pp. 887-888 (1991, Lea & Febiger, Malvern, Pa.).

Applicants have found that processes using thermal treatment may resultin a substantial loss in protease inhibitory activity and, thus, shouldbe used with caution. For example, applicants have found that heatingsoymilk to 100° C. for only 10 minutes reduced the trypsin inhibitoryactivity of the soymilk from 86% (when maintained at 4° C.) to 46%.Applicants have found that heating soymilk can also result in a changeof the color or odor of the soybean product.

Topical Compositions

The topical compositions useful in the present invention involveformulations suitable for topical application to skin In one embodiment,the composition comprises the soy product and a cosmetically-acceptabletopical carrier. In one embodiment, the cosmetically-acceptable topicalcarrier is from about 50% to about 99.99%, by weight, of the composition(e.g., from about 80% to about 95%, by weight, of the composition.

The compositions may be made into a wide variety of product types thatinclude but are not limited to lotions, creams, gels, sticks, sprays,shaving creams, ointments, cleansing liquid washes and solid bars,shampoos, pastes, powders, mousses, shaving creams, wipes, patches, naillacquers, wound dressing and adhesive bandages, hydrogels, films andmake-up such as foundations, mascaras, and lipsticks. These producttypes may comprise several types of cosmetically acceptable topicalcarriers including, but not limited to solutions, emulsions (e.g.,microemulsions and nanoemulsions), gels, solids and liposomes. Thefollowing are non-limitative examples of such carriers. Other carrierscan be formulated by those of ordinary skill in the art.

The topical compositions useful in the present invention can beformulated as solutions. Solutions typically include an aqueous solvent(e.g., from about 50% to about 99.99% or from about 90% to about 99% ofa cosmetically acceptable aqueous solvent).

Topical compositions useful in the subject invention may be formulatedas a solution comprising an emollient. Such compositions preferablycontain from about 2% to about 50% of an emollient(s). As used herein,“emollients” refer to materials used for the prevention or relief ofdryness, as well as for the protection of the skin. A wide variety ofsuitable emollients are known and may be used herein. Sagarin,Cosmetics, Science and Technology, 2nd Edition, Vol. 1, pp. 32-43 (1972)and the International Cosmetic Ingredient Dictionary and Handbook, eds.Wenninger and McEwen, pp. 1656-61, 1626, and 1654-55 (The Cosmetic,Toiletry, and Fragrance Assoc., Washington, D.C., 7^(th) Edition, 1997)(hereinafter “ICI Handbook”) contains numerous examples of suitablematerials.

A lotion can be made from such a solution, Lotions typically comprisefrom about 1% to about 20% (e.g., from about 5% to about 10%) of anemollient(s) and from about 50% to about 90% (e.g., from about 60% toabout 30%) of water.

Another type of product that may be formulated from a solution is acream. A cream typically comprises from about 5% to about 50% (e.g.,from about 10% to about 20%) of an emollient(s) and from about 45% toabout 85% (e.g., from about 50% to about 75%) of water.

Yet another type of product that may be formulated from a solution is anointment. An ointment may comprise a simple base of animal or vegetableoils or semi-solid hydrocarbons. An ointment may comprise from about 2%to about 10% of an emollient(s) plus from about 0.1% to about 2% of athickening agent(s). A more complete disclosure of thickening agents orviscosity increasing agents useful herein can be found in Sagarin,Cosmetics, Science and Technology, 2nd Edition, Vol. 1, pp. 72-73 (1972)and the ICI Handbook pp. 1693-1697.

The topical compositions useful in the present invention formulated asemulsions. If the carrier is an emulsion, from about 1% to about 10%(e.g., from about 2% to about 5%) of the carrier comprises anemulsifiers) Emulsifiers may be nonionic, anionic or cationic. Suitableemulsifiers are disclosed in, for example, U.S. Pat. No. 3,755,5607 U.S.Pat. No. 4,421,769, McCutcheon's Detergents and Emulsifiers, NorthAmerican Edition, pp. 317-324 (1986), and the ICI Handbook, pp.1673-1686.

Lotions and creams can be formulated as emulsions. Typically suchlotions comprise from 0.5% to about 5% of an emulsifiers(s). Such creamswould typically comprise from about 1% to about 20% (e.g., from about 5%to about 10%) of an emollient(s); from about 20% to about 80% (e.g.,from 30% to about 70%) of water; and from about 1% to about 10% (e.g.,from about 2% to about 5%) of an emulsifier(s).

Single emulsion skin care preparations, such as lotions and creams, ofthe oil-in-water type and water-in-oil type are well-known in thecosmetic art and are useful in the subject invention. Multiphaseemulsion compositions, such as the water-in-oil-in-water type, asdisclosed in U.S. Pat. Nos. 4,254,105 and 4,960,764, are also useful inthe subject invention. In general, such single or multiphase emulsionscontain water, emollients, and emulsifiers as essential ingredients.

The topical compositions of this invention can also be formulated as agel (e.g., an aqueous gel using a suitable gelling agent (s)). Suitablegelling agents for aqueous gels include, but are not limited to, naturalgums, acrylic acid and acrylate polymers and copolymers, and cellulosederivatives (e.g., hydroxymethyl cellulose and hydroxypropyl cellulose).Suitable gelling agents for oils (such as mineral oil) include, but arenot limited to, hydrogenated butylene/ethylene/styrene copolymer andhydrogenated ethylene/propylene/styrene copolymer. Such gels typicallycomprises between about 0.1% and 5%, by weight, of such gelling agents.

The topical compositions of the present invention can also be formulatedinto a solid formulation (e.g., a wax-based stick, soap bar composition,powder, or a wipe containing powder).

Liposomal formulations are also useful compositions of the subjectinvention. Examples of liposomes are unilamellar, multilamellar, andpaucilamellar liposomes, which may or may not contain phospholipids.Such compositions can be prepared by first combining hesperetin with aphospholipid, such as dipalmitoylphosphatidyl choline, cholesterol andwater according to the method described in Mezei & Gulasekharam,“Liposomes—A Selective Drug Delivery System for the Topical Route ofAdministration; Gel Dosage Form”, Journal of Pharmaceutics andPharmacology, Vol. 34 (1982), pp. 473-474, or a modification thereof.Epidermal lipids of suitable composition for forming liposomes may besubstituted for the phospholipid. The liposome preparation may thenincorporated into one of the above carriers (e.g., a gel or anoil-in-water emulsion) in order to produce the liposomal formulation.

Other compositions and pharmaceutical uses of topically appliedliposomes are described in Mezei, M., “Liposomes as a Skin Drug DeliverySystem”, Topics in Pharmaceutical Sciences (D. D. Breimer and P.Speiser, eds.), Elsevier Science Publishers P. V., New York, N.Y., 1985,pp. 345-358, PCT Patent Application No. WO96/31194 and U.S. Pat. No.5,260,065.

The topical compositions useful in the subject invention may contain, inaddition to the aforementioned components, a wide variety of additionaloil-soluble materials and/or water-soluble materials conventionally usedin compositions for use on skin, hair, and nails at theirart-established levels.

Additional Cosmetically Active Agents

In one embodiment, the topical composition further comprises anothercosmetically active agent in addition to the legume product. What ismeant by a “cosmetically active agent” is a compound that has a cosmeticor therapeutic effect on the skin, hair, or nails, e.g., lighteningagents, darkening agents such as self-tanning agents, anti-acne agents,shine control agents, anti-microbial agents, anti-inflammatory agents,anti-mycotic agents, anti-parasite agents, external analgesics,sunscreens, photoprotectors, antioxidants, keratolytic agents,detergents/surfactants, moisturizers, nutrients, vitamins, energyenhancers, anti-perspiration agents, astringents, deodorants, hairremovers, firming agents, anti-callous agents, and agents for hair,nail, and/or skin conditioning.

In one embodiment, the agent is selected from, but not limited to, thegroup consisting of hydroxy acids, benzoyl peroxide, sulfur resorcinol,ascorbic acid, D-panthenol, hydroquinone, octyl methoxycinnimate,titanium dioxide, octyl salicylate, homosalate, avobenzone,polyphenolics, carotenoids, free radical scavengers, spin traps,retinoids such as retinol and retinyl palmitate, ceramides,polyunsaturated fatty acids, essential fatty acids, enzymes, enzymeinhibitors, minerals, hormones such as estrogens, steroids such ashydrocortisone, 2-dimethylaminoethanol, copper salts such as copperchloride, peptides containing copper such as Cu:Gly-His-Lys, coenzymeQ10, peptides such as those disclosed in PCT Patent ApplicationWO00/15138, lipoic acid, amino acids such a proline and tyrosine,vitamins, lactobionic acid, acetyl-coenzyme A, niacin, riboflavin,thiamin, ribose, electron transporters such as NADH and FADH2, and otherbotanical extracts such as aloe vera, and derivatives and mixturesthereof. The cosmetically active agent will typically be present in thecomposition of the invention in an amount of from about 0.001% to about20% by weight of the composition, e.g., about 0.01% to about 10% such asabout 0.1% to about 5%.

Examples of vitamins include, but are not limited to, vitamin A, vitaminSs such as vitamin B3, vitamin B5, and vitamin B12, vitamin C, vitaminK, and vitamin E and derivatives thereof.

Examples of hydroxy acids include, but are not limited, to glycolicacid, lactic acid, malic acids salicylic acid, citric acid, and tartaricacid. See, e.g., European Patent Application No. 273,202.

Examples of antioxidants include, but are not limited to, water-solubleantioxidants such as sulfhydryl compounds and their derivatives (e.g.,sodium metabisulfite and N-acetyl-cysteine), lipoic acid anddihydrolipoic acids resveratrol, lactoferrin, and ascorbic acid andascorbic acid derivatives (e.g., ascorbyl palmitate and ascorbylpolypeptide). Oil-soluble antioxidants suitable for use in thecompositions of this invention include, but are not limited to,butylated hydroxytoluene, retincids (e.g., retinol and retinylpalmitate), tocopherols (e.g., tocopherol acetate), tocotrienols, andubiquinone. Natural extracts containing antioxidants suitable for use inthe compositions of this invention, include, but not limited to,extracts containing flavonoids and isoflavonoids and their derivatives(e.g., genistein and diadzein), extracts containing resveratrcl and thelike.

Examples of such natural extracts include grape seed, green tea, pinebark, and propolis Other examples of antioxidants may be found on pages1612-13 of the ICI Handbook.

Other Materials

Various other materials may also be present in the compositions usefulin the subject invention. These include humectants, proteins andpolypeptides, preservatives and an alkaline agent. Examples of suchagents are disclosed in the ICI Handbook, pp. 1650-1667. Thecompositions of the present invention may also comprise chelating agents(e.g., EDTA) and preservatives (e.g., parabens). Examples of suitablepreservatives and chelating agents are listed in pp. 1626 and 1654-55 ofthe ICI Handbook. In addition, the topical compositions useful hereincan contain conventional cosmetic adjuvants, such as dyes, opacifiers(e.g., titanium dioxide) pigments, and fragrances.

Mineral Water

The legume product (e.g., soymilk) and compositions of the presentinvention may be prepared using a mineral water in one embodiment, themineral water has a mineralization of at least about 200 mg/L (e.g.,from about 300 mg/L to about 1000 mg/L). In one embodiment, the mineralwater comprises at least about 10 mg/L of calcium and/or at least about5 mg/L of magnesium.

The composition and formulations containing such compositions of thepresent invention may be prepared using methodology that is well knownby an artisan of ordinary skill.

Example 1 Gamma Irradiation of Legume Product

Applicants have found that soymilk powder prior to any antimicrobialprocessing such as gamma irradiation has high levels microbial content,ranging from up to 50,000 cfu per gram. Such products were also found tohave detectable levels of objectionable microbial content, such as fecalstreptococci, at levels up to 20,000 cfu per gram.

with this substrate (provided in kit), then made to a final workingconcentration of 10 microgram/ml in digestion buffer. Followingincubation of the trypsin, with or without the test material, with theBODIPY fluorescent casein substrate at room temperature for one hour,fluorescence was measured (excitation 485 nm/emission 530 nm) on aSpectraMax® Gemini microtiter plate reader (Molecular DevicesCorporation, Sunnyvale, Calif.) using Softmax® Pro 3.0 software(Molecular Devices Corporation). Each experiment was performed in threereplicates and was repeated twice.

This assay was performed on soy products processed seven different waysExample A was soybeans ground into powder (Sunlight Foods Corporation,Taipei County, Taiwan, R.O.C.). Example B was soybean powder of ExampleA exposed to about 8-15 kGy of gamma irradiation. Example C was soybeanpowder in which the oil in the soybean powder was removed by extraction(Soyafluff® 200W from Central Soya Company, Inc., Fort Weyne, Ind.).Example D was soymilk powder made with dehulled soybeans and water thatwas subsequently filtered and heated and spray dried (Devansoy Farms,Carroll, Iowa) and exposed to between about 7-9 kGy of gammairradiation. Example E was soymilk powder obtained by mixing soy beansand water, heating the mixture overnight, and adding 1,3-butylene glycolto the mixture (Flavosterone SB from Ichimaru Pharcos Co., Ltd, GifuJapan). Example F was soymilk powder obtained by mixing soy beans andwater, heating the mixture overnight, and subsequently adding ethanol tothe mixture (Flavosterone SE from Ichimaru Pharcos Co., Ltd, GifuJapan). Example G was an extract of soy proteins (Vegeseryl HGP LS 8572from Laboratories Serobiologiques S.A., Pulnoy, France).

These soy products were compared to Soy Trypsin Inhibitor (STI) (Sigma).

The percent inhibition of trypsin cleavage of the substrate by thedifferent soy preparations was calculated using Microsoft Excel™ and isreported In Table 1.

TABLE 1 % Inhibition of Tested Product Concentration Trypsin STI 0.0143.0 STI 0.1 76.1 Example A 0.01 32.8 Example A 0.1 67.1 Example B 0.0131.5 Example B 0.1 67.2 Example C 0.01 22.7 Example C 0.1 36.2 Example D0.01 8.92 Example D 0.1 17.4 Example E 0.01 7.83 Example E 0.1 10.8Example F 0.01 4.87 Example F 0.1 5.99 Example G 0.1 6.85

As shown in Table 1, STI can inhibit trypsin-induced cleavage in a doseresponse manner. Example A, which is soybean powder, also significantlyinhibited trypsin activity. Further gamma irradiation of the soybeanpowder (i.e., Example B), while reducing the microbial content of thesoybean powder, unexpectedly did not significantly impact the trypsininhibition activity of the soybean powder. The heat and/or extractionprocessing of Examples C-C, however, did significantly reduce thetrypsin inhibitory activity of the soybean powder.

Example 3 Thiol Retention Activity of Legume Product

The ability of soy powder to prevent smoke-induced Loss of thiols wasevaluated in normal human dermal fibroblasts (Clonetics, San Diego,Calif.). Thiols, chiefly glutathione, are part of the endogenouscellular antioxidant defense system. Glutathione serves as a redoxbuffer, thereby, maintaining the balance between oxidants andantioxidants. Glutathione is also the preferred substrate for severalenzymes such as the glutathione peroxidases (decomposing peroxides) andthe glutathione-S-transferases (a major group of detoxificationenzymes). See, A. Meister, Cancer Res. 54:1969s-1975s (1994).

Cutaneous antioxidants (both enzymatic and non-enzymatic), includingglutathione, are depleted after UV or ozone exposure. See, M. J. Connorand L. A. Wheeler, Photochem. Photobiol. 46:239-246 (1987) and R. M.Tyrrell and M. Pidoux, Photochem. Photobiol. 47:405-412 (1988). In cellculture models, low intracellular glutathione (GSH) levels lead to ahigher UV radiation sensitivity. Topical application of cysteinederivatives on rat skin has been shown to protect against UVradiation-induced photodamage; this benefit was correlated with anincrease in GSH synthesis. See, L. T. van den Broeke and G. M. J.Beijersbergen van Henegouwen, J. Photochem. Photobiol. B Biol. 27:61-65(1995); K. Hanada, et al., J. Invest. Dermatol. 108:727-730 (1997); andD. P. T. Steenvoorden, et al., Photochem Photobiol. 67:651-656 (1998).Consequently, glucothione is a major endogenous antioxidant, highlyresponsive against environmental challenges, able to regulate the toneand the wrinkling of skin, as well as treat external aggression.

In this experiment, normal human neonatal dermal fibroblasts seeded in24-well format Transwell inserts to (Corning Costar, Cambridge, Mass.)were incubated with media containing various concentrations of soymilkpowder (gamma irradiated at about 5 kGy) for 24 hours prior to exposurewith either placebo (mock) or cigarette smoke (1 cigarette, BASIC FullFlavor 100's cigarettes, Philip Morris, Richmond, Va.) for 10 minutes.

Prior to smoke exposure, the medium above the inserts containing the soyproduct was removed, and the cells were washed 3 times with Dulbecco'sPhosphate-Buffered Saline (Life Technologies, Gaithersburg, Md.) beforebeing smoke-exposed with only media below the inserts. Immediately afterexposure, the cells were incubated for another 24-hour period with theprevious medium. The cells were washed again, 5 times with Dulbecco'sPhosphate-Buffered Saline, and intracellular thiols were then measuredby adding 60 μM monobromobimane (Molecular Probes, Eugene, Oreg., USA)to the cells and incubating at 37° C. for 30 minutes before thefluorescence reading. In the presence of thiols, the monobromobimanebecomes fluorescent. This fluorescence was measured using a CytoFluor®Fluorescence Plate Reader (PerSeptive Biosystems, Framingham, Mass.,USA) set with the following filter combination: excitation at 360 nm andemission at 460 nm.

The results of this experiment are set-forth in Table 2.

TABLE 2 Thiols (Percent of Thiols Soymilk Powder contained in Noconcentration Smoke Group; (weight %) Mean ± S.E.M.) No Smoke 0  100 ±6.71 Smoke (10 min.) 0 65.38 ± 7.16  0.5 91.24 ± 14.25 1 95.39 ± 4.52  2106.92 ± 17.06 

These results indicate that gamma irradiated soymilk powder surprisinglyafforded a protection against smoke-induced loss of thiols (datarepresent the mean±Standard of the mean of replicates from 3 independentexperiments).

It is understood that while the invention has been described inconjunction with the detailed description thereof, that the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the claims.

1. A composition for topical application comprising: (a) a soy producthaving a trypsin inhibitory activity of at least about 15% and amicrobial content less than about 1000 cfu per gram; and (b) acosmetically-acceptable topical carrier.
 2. A composition of claim 1,said soy product having an objectionable microbial count less than about150 cfu per gram.
 3. A composition of claim 1, said soy product having amicrobial content of less than about 100 cfu per gram.
 4. A compositionof claim 1, wherein said soy product has a thiol retention activity ofat least about 75%.
 5. A composition of claim 1, wherein said soyproduct has been exposed to gamma irradiation.
 6. A composition of claim1, wherein said soy product is soymilk powder.
 7. A composition of claim1, wherein said soy product is soybean powder.
 8. A composition of claim1, wherein said composition further comprises vitamin A, vitamin B3,vitamin B5, vitamin B12, vitamin C, vitamin K, vitamin E, andderivatives thereof.
 9. A composition of claim 1, wherein saidcomposition further comprises 2-diemthylaminoethanol.
 10. A compositionof claim 1, wherein said composition further comprises salicylic acid,lactic acid, or glycolic acid.
 11. A composition of claim 1, whereinsaid composition further comprises N-acetyl-cysteine.
 12. A compositionfor topical application comprising: (a) a soy product having a trypsininhibitory activity of at least about 50% and a microbial content lessthan about 10,000 cfu per gram; and (b) a cosmetically-acceptabletopical carrier.
 13. A composition of claim 12, said soy product havingan objectionable microbial count less than about 150 cfu per gram.
 14. Acomposition of claim 12, said soy product having a microbial content ofless than about 1000 cfu per gram.
 15. A composition of claim 12, saidsoy product having no detectable objectionable microbial content pergram.
 16. A composition of claim 12, wherein said soy product has beenexposed to gamma irradiation.